Comparison of growth limits of Listeria monocytogenes in milk,broth and cheese

Aim: To determine growth initiation differences of Listeria monocytogenes between a cheesemaking context, milk and tryptic soy broth (TSB). Methods and Results: A laboratory‐scale cheese was made with a mix of two strains of L. monocytogenes at four initial pH values, five water activity (a_w) values and two contamination levels at 30°C. Counts of L. monocytogenes were determined at time 0 and after 8 h of cheese manufacture. Milk and TSB at the same pH and a_w conditions were inoculated with the L. monocytogenes mix in multi‐well plates. Growth was determined by plating each well onto Agosti & Ottaviani Listeria Agar after 8 h of incubation at 30°C. Each condition was repeated six times, and growth initiation probability was modelled with logistic regression models. Growth initiation boundaries were obtained for each matrix type. The results showed that the growth limits were matrix dependent. In the three matrix types, a_w was the most important factor affecting the probability of growth initiation. Contamination level affected growth TSB and cheesemaking conditions. Conclusions: The interface wideness and position in cheese, milk and TSB were dissimilar, indicating that the use of models evaluated in TSB or milk could not be used to predict the behaviour of L. monocytogenes under cheesemaking conditions. Significance and Impact of the Study: Predictive models generated in liquid media are not necessarily adaptable to solid food, and the generation of real food models is necessary.     

Glutamate Decarboxylase-Mediated Nisin Resistance in Listeria monocytogenes

Analysis of a complete set of glutamate decarboxylase (gad) mutants of Listeria monocytogenes strain LO28 (ΔgadD1, ΔgadDT1, ΔgadD2, ΔgadT2, and ΔgadD3 mutants) revealed that the ΔgadD1 mutant is impaired in its ability to tolerate exposure to both sublethal and lethal levels of the lantibiotic nisin. gadD1 is strain variable and is found only in approximately 50% of L. monocytogenes strains. Growth and survival experiments revealed that possession of gadD1 correlates with a higher degree of tolerance to nisin. Significantly, a similar finding using a gadB mutant of L. lactis IL1403 implies that this may be a general phenomenon in Gram-positive bacteria. Our findings thus suggest that the specific inhibition of GAD activity or a reduction in the levels of free glutamate may prevent the growth of otherwise resistant GAD⁺ bacteria in foods where low pH and/or nisin is used as a preservative.Listeria monocytogenes is a food-borne pathogen that is the causative agent of listeriosis, an opportunistic infection associated with high rates of morbidity and mortality (18). The microorganism has also been the cause of significant commercial losses, being responsible for 71% of all recalls of food products due to bacterial contamination in the United States between 1993 and 1998 (25). The ubiquitous nature of L. monocytogenes, together with its ability to tolerate a variety of environmental extremes, including high salt concentrations and low pH, and the ability to grow at refrigeration temperatures, makes control of the bacterium in foods difficult (10). Hence it is not altogether surprising that the food industry invests considerable effort into developing strategies to prevent the survival and growth of this pathogen. One such strategy involves the utilization of bacteriocins. Bacteriocins are antimicrobial peptides produced by one bacterium that inhibit the growth of other bacteria and have been used as “natural” preservatives to control undesirable microbiota in food (5). The most extensively studied bacteriocin is nisin A (here referred to as nisin), a 34-amino-acid class I bacteriocin (lantibiotic) produced by Lactococcus lactis strains that is currently approved for use in foods in over 50 countries. Nisin functions by binding lipid II, an essential precursor of cell wall peptidoglycan biosynthesis. Binding to lipid II also facilitates the formation of pores within the cytoplasmic membrane leading to the release of ATP and other small cytoplasmic contents, resulting in depolarization of the membrane potential and ultimately cell death (13).The molecular mechanisms employed by L. monocytogenes to cope with nisin are poorly understood. To date, loci that have been implicated in nisin tolerance include the alternative stress sigma factor SigB, the class three stress gene regulator CtsR, the two-component systems LisRK and HK1027, and a penicillin binding protein, Pbp (2, 6, 11, 15, 21). In addition, several studies have uncovered a link between the acid stress response of L. monocytogenes and nisin resistance (3, 17, 24). Several systems are employed by L. monocytogenes to withstand low pH stress, but the glutamate decarboxylase (GAD) system is probably the most important (an overview of the GAD system is in Fig. ​Fig.1).1). Mutation of specific gad genes renders cells exquisitely sensitive to ex vivo porcine and synthetic human gastric fluid and significantly impairs growth and survival in low-pH foods (4, 7, 8). Given the link between acid and nisin resistance phenotypes, the present study was initiated in order to investigate the contribution, if any, of gad genes to the nisin tolerance of L. monocytogenes.Open in a separate windowFIG. 1.An overview of the L. monocytogenes glutamate decarboxylase (GAD) system. L. monocytogenes possesses five gad genes. gadD1, gadD2, and gadD3 encode decarboxylases which catalyze the conversion of glutamate to γ-amino butyrate (GABA) and carbon dioxide (CO₂). gadT1 and gadT2 encode glutamate-GABA antiporters. Nisin functions by binding to lipid II, an essential precursor of cell wall peptidoglycan synthesis. Binding to lipid II facilitates the formation of pores within the cytoplasmic membrane leading to the release of ATP and may ultimately result in cell death. We suggest that under certain conditions gadD1 may contribute to intracellular ATP pools and hence tolerance of nisin.     

Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes

The food-borne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates, reaching up to 25 to 30%. Listeria is a hardy organism, and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semipurified preparations (SPP) containing either nisin A or an enhanced bioengineered derivative, nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol, and trans-cinnamaldehyde, to control Listeria monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing SPP (25 μg/ml) with thymol (0.02%), carvacrol (0.02%), or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil-nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V-carvacrol or nisin V-cinnamaldehyde combinations was twice that of the equivalent nisin A-essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural, and effective means to enhance food safety by inhibiting food-borne pathogens such as L. monocytogenes.     

Antilisterial activity of lactose monolaurate in milk,drinkable yogurt and cottage cheese

Lactose monolaurate (LML) was previously found to be an antimicrobial against Listeria monocytogenes in culture medium at concentrations between 3 and 5 mg ml^?1. In this study, the microbial inhibitory activity of LML in dairy products inoculated with a 5‐strain cocktail of clinical isolates of L. monocytogenes was investigated. Addition of LML at a concentration of 5 mg ml^?1 resulted in 4·4, 4·0 and 4·2 log reductions in 0·5% fat, 1% fat and 3·25% fat milks, respectively; 4·1, 4·4, and 3·5 log reductions in nonfat, 1% fat, and 1·5% fat yogurts, respectively; and 4·0 log reductions in both nonfat and 2% fat cottage cheese. The inhibitory effect of LML was only observed at 37°C and not 5°C. Experiments suggest that both the lauric acid and the esterified lactose moiety of LML play roles in the growth inhibition.

Significance and Impact of the Study

A novel sugar ester, lactose monolaurate, inhibited the growth of a five‐strain cocktail of Listeria monocytogenes in milk, yogurt and cottage cheese. This is the first report of the use of a sugar ester to inhibit the growth of Listeria in food systems.     

Adaptation of Listeria monocytogenes in a simulated cheese medium: effects on virulence using the Galleria mellonella infection model

The aim of this study was to evaluate the effect of the acid and salt adaptation in a cheese‐based medium on the virulence potential of Listeria monocytogenes strains isolated from cheese and dairy processing environment using the Galleria mellonella model. Four L. monocytogenes strains were exposed to a cheese‐based medium in conditions of induction of an acid tolerance response and osmotolerance response (pH 5·5 and 3·5% w/v NaCl) and injected in G. mellonella insects. The survival of insects and the L. monocytogenes growth kinetics in insects were evaluated. The gene expression of hly, actA and inlA genes was determined by real‐time PCR. The adapted cells of two dairy strains showed reduced insect mortality (P < 0·05) in comparison with nonadapted cells. Listeria monocytogenes Scott A was the least virulent, whereas the cheese isolate C882 caused the highest insect mortality, and no differences (P > 0·05) was found between adapted and nonadapted cells. The gene expression results evidenced an overexpression of virulence genes in cheese‐based medium, but not in simulated insect‐induced conditions. Our results suggest that adaptation to low pH and salt in a cheese‐based medium can affect the virulence of L. monocytogenes, but this effect is strain dependent.

Significance and Impact of the Study

In this study, the impact of adaptation to low pH and salt in a cheese‐based medium on L. monocytogenes virulence was tested using the Wax Moth G. mellonella model. This model allowed the differentiation of the virulence potential between the L. monocytogenes strains. The effect of adaptation on virulence is strain dependent. The G. mellonella model revealed to be a prompt method to test food‐related factors on L. monocytogenes virulence.     

Dietary Restraint and Control Over “Wanting” Following Consumption of “Forbidden” Food

Eating behavior can be influenced by the rewarding value of food, i.e., “liking” and “wanting.” The objective of this study was to assess in normal‐weight dietary restrained (NR) vs. unrestrained (NU) eaters how rewarding value of food is affected by satiety, and by eating a nonhealthy perceived, dessert‐specific food vs. a healthy perceived, neutral food (chocolate mousse vs. cottage cheese). Subjects (24NR age = 25.0 ± 8.2 years, BMI = 22.3 ± 2.1 kg/m²; 26NU age = 24.8 ± 8.0 years, BMI = 22.1 ± 1.7 kg/m²) came to the university twice, fasted (randomized crossover design). Per test‐session “liking” and “wanting” for 72 items divided in six categories (bread, filling, drinks, dessert, sweets, stationery (placebo)) was measured, before and after consumption of chocolate mousse/cottage cheese, matched for energy content (5.6 kJ/g) and individual daily energy requirements (10%). Chocolate mousse was liked more than cottage cheese (P < 0.05). After consumption of chocolate mousse or cottage cheese, appetite and “liking” vs. placebo were decreased in NR and NU (P < 0.03), whereas “wanting” was only decreased in NR vs. NU (P ≤ 0.01). In NR vs. NU “wanting” was specifically decreased after chocolate mousse vs. cottage cheese; this decrease concerned especially “wanting” for bread and filling (P < 0.05). To conclude, despite similar decreases in appetite and “liking” after a meal in NR and NU, NR decrease “wanting” in contrast to NU. NR decrease “wanting” specifically for a nonhealthy perceived, “delicious,” dessert‐specific food vs. a nutritional identical, yet healthy perceived, slightly less “delicious,” “neutral” food. A healthy perceived food may thus impose greater risk for control of energy intake in NR.     

Divergent Evolution of the Activity and Regulation of the Glutamate Decarboxylase Systems in Listeria monocytogenes EGD-e and 10403S: Roles in Virulence and Acid Tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GAD_i), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GAD_i system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GAD_i/GAD_e). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GAD_i but not GAD_e the results indicate that the GAD_i system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.     

c‐di‐AMP modulates Listeria monocytogenes central metabolism to regulate growth,antibiotic resistance and osmoregulation

Cyclic diadenosine monophosphate (c‐di‐AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall‐active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β‐lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl‐CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100‐fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c‐di‐AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down‐stream enzymes suppressed ΔdacA phenotypes. These data suggested that c‐di‐AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild‐type bacteria.     

Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli

Aims: To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance. Methods and Results: The organic solvent tolerance of ΔbetT, ΔproV, ΔproP or ΔputP single‐gene knockout mutants of E. coli K‐12 strain was examined. Among these mutants, the organic solvent tolerance of the ΔproV mutant remarkably increased compared with that of the parent strain. It has been known that a marR mutation confers tolerance on E. coli to organic solvents. A ΔproV and ΔmarR double‐gene mutant was more tolerant to organic solvents than the ΔproV or ΔmarR single‐gene mutant. The n‐hexane amount accumulated in E. coli cells was examined after incubation in an n‐hexane‐aqueous medium two‐phase system. The intracellular n‐hexane level in the ΔproV and ΔmarR double‐gene mutant was kept lower than those of the parent strain, ΔproV mutant and ΔmarR mutant. Conclusions: The organic solvent tolerance level in E. coli highly increased by dual disruption of proV and marR. Significance and Impact of the Study: This study suggests a new strategy for increasing the organic solvent tolerance level in E. coli to improve the usability of the whole‐cell biocatalysts in two‐phase systems employing organic solvents.     

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi‐functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N‐deacetylase PgdA and the peptidoglycan O‐acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA‐dependent and OatA‐independent. Protein‐protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB‐like cell division protein and factors important for escape from the host immune system.     

Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at low pH

The glutamate decarboxylase (GAD) system is critical to the survival of Listeria monocytogenes LO28 at low-pH stress (相似文献   

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Fibronectin‐binding protein,FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA‐deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild‐type L. monocytogenes. FbpA did not affect the amounts of various virulence‐determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild‐type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.     

Natural Variation in Susceptibility of Listeria Strains to Class IIa Bacteriocins

Thirty-one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely, enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern in which all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class IIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives. Received: 22 October 1999 / Accepted: 15 December 1999